Indican and suspensions and gels thereof and their uses

ABSTRACT

Indican, a polysaccharide comprising (1→3) glucose, (1→4) mannose, (1→4) rhamnose and (1→3 or 4) -0-(1-carboxyethyl)-rhamnose units in a molar ratio of about 2:1:1-2:1 respectively; containing 12-15% by weight acetyl units; [α]D 20  about -61°; having principle absorption bands in the infra red band at 3390, 1735, 1615, 1375, 1250 and 1050 cm -1  ; a solubility of at least 1% by weight in methanol and in ethylene glycol, and an inherent viscosity of about 33.5 dl/g. has useful thixotropic properties and provides a method of modifying the viscosity of a liquid by incorporating indican therein. The liquid may be aqueous or organic solvent-based. Thickened and stabilized products and a method of preparing indican by culture of an indican-producing microorganism are also provided.

This invention relates to the use of a microbial polysaccharide as asuspending, emulsifying or gelling agent.

Various polysaccharides are known which have the ability to modify theviscosity of an aqueous medium in order to confer pseudoplasticity.Pseudoplasticity, and also thixotropy, and a high yield stress arevaluable characteristics in many fields, for example in emulsion paintmanufacture, in agricultural sprays, in well-drilling fluids and so on.In this specification, the term thixotropy is intended to mean thecharacteristic of lowering of viscosity of liquids when subjected toshear. Pseudoplasticity is regarded as the particular case where theregaining of viscosity is extremely rapid.

Xanthan gum exhibits some activity in this respect, particularly whenadmixed with galactomannan gums, but its viscosity is too rapidlyregained for some purposes.

A microbial polysaccharide which possesses useful characteristics ofthis type is described in U.S. Patent No. 3,960,832. Thispolysaccharide, known as Heteropolysaccharide 7, is derived fromAzotobacter indicus var. myxogenes. This polysaccharide has beenproposed for use in emulsion paints, for example in U.S. Pat. No.3,894,976, and in well-drilling fluids, for example in British Pat. No.1,416,013.

We have now obtained, from a different microbial source, a new microbialpolysaccharide which is chemically different from Heteropolysaccharide 7but which possesses a rheology giving it similar uses, together withother important properties not shared by Heteropolysaccharide 7.

The polysaccharide according to the present invention, is derived from amicroorganism deposited at the American Type Culture Collection underthe No. ATCC 19361 by H. Jensen in 1957. The microorganism wasoriginally deposited under the name Azotobacter lacticogenes. However,in the latest edition of Bergey's Manual of Determinative Bacteriology(8th Edition 1974), A. lacticogenes is no longer recognised as aseparate species, but, together with Beijerinckia lacticogenes andseveral other species is now classified as B. indica. The nomenclatureof the microorganisms in question is thus rather complex and is apt tobe misleading. We have compared the microorganism ATCC 19361 with thetype strain of B. indica, deposited as NCIB 8712, and also, forreference, with A. indicus var. myxogenes ATCC 21423. From ourexperiments it is clear that the microorganism deposited as ATCC 19361should, in fact, be classified as a strain of B. indica, and will now bereferred to in this specification as B. indica ATCC 19361. From ourexperiments, it is also clear that A. indicus var. myxogenes ATCC 21423differs significantly from both the other organisms tested. It is indeeda species of Azotobacter rather than Beijerinckia since:

(i) it grows readily in nutrient broth and on nutrient agar;

(ii) it forms a pellicle in most liquid media; and

(iii) it forms a yellow non-water soluble pigment.

Details of the comparison are given later in this specification.

The polysaccharide according to the present invention is thepolysaccharide obtained by culture of B. indica ATCC 19361 in a nutrientmedium and then subsequently precipitating the polysaccharide produced,e.g. by addition of an alcohol such as isopropyl alcohol or by renderingthe medium acid. The invention also comprises a substantially identicalpolysaccaride if produced from a different microbial source. Thispolysaccharide will, for convenience, be referred to hereinafter asindican.

The type strain of B. indica is B. indica Starket and De, ATCC 9039which is identical to NCIB 8712. This strain was reported by Quinnell etal. (Can. J. Microbiol. 3 (1957) 277) to produce a polysaccharidecomprising units of glucose, glucuronic acid and a heptose. Haug andLarsen (Acta. Chem. Scand. 24 (1970) 1855) reported that the uronic acidwas, in fact, guluronic acid. Lopex and Backing (Microbiol. Espana 2153) showed that B. indica strain Hawaii-2produces a polysaccharide whichcomprises units of glucose, galactose and mannose and glucuronic andgalacturonic acid, and this combination is cited by Bergey (8th Edition)for the polysaccharide produced by Beijerinckia.

Sowa, in a Ph.D thesis at Queens University, Canada, in 1962, indicatedthat a strain of "B.lacticogens" produced a polysaccharide comprisingunits of glucose, uronic acid, rhamnose and mannose (in order ofdecreasing concentration). In contrast, indican comprises units ofglucose, mannose, rhamnose and a carboxyethyl rhamnose; whileHeteropolysaccharide 7, derived from Azotobacter indicus var myxogenes,comprises units of glucose, rhamnose and a uronic acid. Indican is alsosoluble in methanol at 1% by weight, whereas Heteropolysaccharide 7 isquite insoluble in methanol at that concentration. This solubility innon-aqueous solvents gives indican additional uses of considerableinterest.

According to the present invention there is provided indican, apolysaccharide comprising (1→3) glucose, (1→4) mannose, (1→4) rhamnoseand (1→3 or 4) 0-(1-carboxyethyl)-rhamnose units in a molar ratio ofabout 2:1:1-2:1 respectively; containing 12-15% by weight acetyl units;[α]_(D) ²⁰ about -61°; having principle absorption bands in the infrared band at 3390, 1735, 1615, 1375, 1250 and 1050 cm⁻¹ ; a solubility ofat least 1% by weight in methanol and in ethylene glycol, and aninherent viscosity of about 33.5 dl/g, and especially substantiallycell-free indican obtained from a culture of Beijerinckia indica ATCC19361.

According to the present invention, there is also provided a method forpreparing indican comprising culturing an indican-producing strain ofmicroorganism in a nutrient medium therefor, and isolating the indicanformed from the culture medium. The microorganism is, for example, astrain of B.indica, especially B.indica ATCC 19361.

According to the present invention, there is also provided a method ofmodifying the viscosity of a liquid, especially a polar liquid, byincorporating therein an effective amount of indican.

According to one feature of the present invention, there is provided amethod of modifying the viscosity of an aqueous fluid, particularlyrendering it pseudoplastic, by incorporating therein an effective amountof indican.

According to another feature of the present invention, there is provideda method of suspending a particulate solid in an alcoholic medium byincorporating in the medium an effective amount of indican.

Another feature of the present invention is a method for gelling thecooking liquor around a meat product or the like by incorporatingtherein an effective amount of indican.

According to another feature of the invention, there is provided athixotropic, thickened liquid containing Indican substantially free fromcells of B. indica, especially an aqueous liquid.

According to another feature of the invention, there is provided asuspension of a particulate solid in an alcoholic medium containingindican. In particular, there is provided a suspension of xanthan gum inan alcoholic medium containing indican. Alginate and CMC can also besuspended.

According to another feature of the invention, there is provided anaqueous emulsion of a hydrophobic liquid, in particular an oil orpetroleum product, or of a water-in-oil type of emulsion, containingindican as an emulsifying agent.

According to another feature of the invention, there is provided acanned food product, particularly meat or a meat-like product, in ajelly containing indican.

According to another feature of the invention, there is provided a paintstripper composition containing a paint solvent together with indican asa thickener.

According to another feature of the invention, there is provided aflexible film of indican formed by evaporating the solvent from a thinlayer of an indican solution. Such a film has uses in a wide range offields from painting to metal foil manufacture.

In any of the above uses, the amount of Indican will vary depending onthe results required. In general, an amount of 0.01% to 2% by weight canbe used. For emulsification, gelling and suspension of solids inalcohols, a proportion of 0.1 to 0.5%, particularly about 0.25% byweight is advantageous.

The ability of indican to thicken and render pseudoplastic an aqueousmedium means that the polysaccharide is of particular interest as athickening agent in a number of different applications. One particularlyimportant field is that of aqueous emulsion paints, which are desirablyformulated in a non-drip form. In general, an amount of from 0.01 to 2%by weight of indican can be incorporated in the aqueous emulsion,together with the conventional latex of film-forming resin and pigments.Other components of the paint may be the conventional extenders,anti-foaming agents, dispersion agents, thermal stabilisers andpreservatives. The latex may comprise a styrene-butadiene copolymer, apolystyrene, a polyacrylate or polyvinylacetate emulsion, all of whichare conventional in paint technology.

Another use for the thickening effect is in well-drilling fluids basedon water or brine. In this case, the ability to thin under applied shearenables the fluid to lubricate at areas of high shear, while in areas oflow shear the higher viscosity and gel strength enable drill clays andweighting agents to be entrained and suspended. The emulsifying effectof the polysaccharide also enables the oil to be emulsified with water.

Indican may also be used in water flooding, in secondary oil recovery,either in place of or in combination with xanthan gum.

Pseudoplasticity or thixotropy is also an advantage in aqueous sprays,for example of herbicides and pesticides used in agriculture. Athixotropic fluid will be temporarily thinned on spraying but willthicken after contact with leaf surfaces etc. to prevent run-off.

Other uses in aqueous media include stabilisation of aqueous suspensionsof pigments and minerals in ceramic glazes, in textile printing pastesand in many other uses where conventional thickening and thixotropicagents are used.

A particularly noteworthy feature of indican is its relatively highsolubility in lower alkanols and aqueous alcoholic systems. Oneparticularly important utility provided by this solubility is asfollows. Xanthan gum is now widely used in drilling and water floodingoperations in the oil industry in the form of dilute aqueous solutions.The dry xanthan gum is in the form of a powder which is very difficultto disperse in water. Concentrated aqueous solutions would gel and wouldbe unsuitable. Two main solutions to this problem are in current use.Firstly, the xanthan is suspended as a slurry in liquid paraffin oranother water immiscible solvent, together with a suspension stabiliserand an emulsifier. This suspension can be added to the water and theemulsifier disperses the water immiscible carrier evenly. The resultingxanthan solution, however, is opaque and contains emulsified immisciblesolvent which can be undesirble. The alternative procedure is to suspendthe xanthan in an alcohol such as ethanol with a stabiliser for thesuspension. Alcohol suspensions of this type are easily dispersible inwater to give a homogeneous solution. The stabiliser generally used forthe alcohol suspensions is a cellulose derivative, for example ahydroxypropyl cellulose, in combination with a gelling agent. An exampleof this type of suspension is given in U.S. Pat. No. 3,894,879. However,the hydroxypropyl cellulose derivative and its gelling agent arerelatively inefficient, even when used at relatively high levels, e.g.about 1%.

According to the present invention, indican can be used to stabilise analcoholic or aqueous alcoholic suspension of xanthan gum in the absenceof a cellulose derivative or gelling agent, at a much lowerconcentration, for example of the order of 0.25% by weight of thesuspension medium. Alcohol suspensions of this type are thixotropic andstable on storage and thus can be easily transported either incontainers or by pump. The suspensions disperse easily in water.

Indican also has uses as an emulsifying agent and thus is of interest instabilising xanthan gum suspensions in liquid paraffin and other waterimmiscible solvents. It is also of particular interest in theemulsification of kerosene with water, for example in herbicidal andpesticidal sprays, where, as explained above, it serves the addedpurpose of preventing run-off.

The property of film formation possessed by indican renders it ofconsiderable interest in any field where a cast polysaccharide film isdesirable. The gum can be dissolved in a volatile solvent such asmethanol methylene chloride mixture and allowed to dry on a smoothsurface. The film can be stripped off as a flexible material. Thesolubility in solvents such as methanol methylene chloride also rendersindican very suitable for use in thickened paint stripper formulations.

Finally, indican possesses the property of dissolving in a solvent suchas water to form a viscous solution/gel, which remains highly viscous onbeing heated and becomes a firm gel on recooling. This property meansthat indican is of particular use in the canning industry, as areplacement for xanthan gum/carob gum mixtures. Chunky meat products,such as pet foods, can be evenly distributed in a jelly, the viscosityof which does not markedly decrease when hot, thus preventing settlingof the contents of the can. In this respect, indican is distinctlydifferent from the reported Heteropolysaccharide 7.

As explained earlier, indican may be produced by culture of B. indicaATCC 19361. A comparison of B. indica ATCC 19361 with the type strain,NCIB 8712 was carried out. As the same time, these two strains werecompared with A. indicus var myxogenes ATCA 21423, the source ofHeteropolysaccharide 7.

                  TABLE 1                                                         ______________________________________                                        Tests used in Comparative Study                                               ______________________________________                                        Cell morphology                                                                            Gram stain; spore stain; acid fast                                            stain; capsules; motility.                                       Colonial morphology                                                                        YM agar; Burk's agar + glucose; Burk's                                        agar + sucrose.                                                  Growth characteristics                                                                     Nutrient broth; YM broth; nutrient                                            agar; optimum temperature; pH range;                                          salt tolerance in YM broth.                                      Biochemical charac-                                                                        Methyl red; catalase; indole formation;                          teristics    urease, arginase; oxidase; amylase;                                           cellulase; gelatin liquefaction;                                              nitrate reduction; H.sub.2 S formation;                                       citrate utilisation; acetylmethyl-                                            carbinol formation (Voges - Proskauer)                           Carbohydrate utili-                                                                        20 carbon sources used                                           sation and acid                                                               production                                                                    Effect of amino acids                                                                      20 amino acids used                                              on pigmentation                                                               and growth                                                                    ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Principal Differences Observed                                                                                  A. indicus                                              B. indica             var.                                                    type strain                                                                              B. indica  myxogenes                                   Test        NCIB 8712  ATCC 19361 ATCC 21423                                  ______________________________________                                        1   Motility    weakly +   Majority strongly +                                                           weakly +                                           2   Growth in E1                                                                              clumps     clumps   chains                                        medium                                                                    3   Urease      -          +        -                                         4   Oxidase     weakly +   +        -                                         5   Amylase     no growth  no growth                                                                              +                                             Starch nut. agar                                                              YM + starch -          -        Weak +                                    6   Carbohydrate                                                                  utilisation                                                                   and acid prodn.                                                               arabinose   +(13 days) +(13 days)                                                                             v. weak +                                                                     (21 days)                                     xylose      - or weak +                                                                              - or weak +                                                                            +                                             maltose     +(21 days) -        +                                             trehalose   - or +     -        +                                             lactose     -          -        +                                             cellobiose  -          -        +                                             melibiose   -          +(21 days)                                                                             +                                             dextran     weak +     -        +                                                         (21 days)                                                         mannitol    +          weak +   -                                         7   Amino acids:                                                                  growth and                                                                    pigment prodn.                                                                DL threonine                                                                              growth +   growth + growth -                                      pigment     cream      cream    yellow                                    8   Nutrient broth:                                                                           -          -        yellow; +;                                    growth                          pellicle                                  9   Nutrient agar:                                                                            -          -        yellow; +;                                    growth                          0.5-2 mm                                  10  pH range                                                                      YM broth    4.6-7.2    4.6-7.2  4.6-7.2                                       Nut. broth  + only at  + only at                                                                              3.9-7.9                                                   7.4-7.9    7.9                                                11  Salt toler- weak at 1% 0.4% +;  + at 1.6;                                     ance %                 0.6% -   weak at 2.0                               ______________________________________                                    

It will be seen that ATCC 19361 bears a close resemblence to NCIB 8712and may be classified as B. indica according to Bergey (8th Edition). Onthe other hand, A. indicus var. myxogenes ATCC 21423 is probablycorrectly classified as a species of Azotobacter because of its readygrowth in nutrient broth and on nutrient agar, its formation of pelliclein most liquid media, and the production of a yellow water-insolublepigment.

DESCRIPTION OF ATCC 19361 Cell Morphology

Size: 0.5-1.2 m by 1.6-3.0 m

Shape: Straight or slightly curved rods. Characteristic large, highlyrefractile lipoid bodies occurring at each end of cell, which persist inaged cultures.

Resting stages: Neither cysts nor spores observed.

Gram reaction: Negative

Acid fast Stain: Negative

Motility: Most negative, a few weakly positive.

    ______________________________________                                        Colonial Morphology                                                           YM Agar       Tenacious gum, cream colonies smaller                           (27° C. 6 days)                                                                      than type strain, circular, entire,                                           domed, opaque, up to 3mm diameter.                              Burk's medium +                                                                             Punctiform colonies less than 1mm                               glucose       diam. circular, entire, domed.                                  (27°  C. 6 days)                                                                     Inoculation point-thick white                                                 tenacious elastic gum.                                          Burk's medium +                                                                             Punctiform colonies, translucent.                               Sucrose       Inoculation point - dense growth                                (27° C. 6 days)                                                                      watery gum                                                      Nutrient Agar No growth                                                       (27° C. 14 days)                                                       Malt Agar     Weak growth, punctiform colonies                                (27° C. 14 days)                                                       Growth Characteristics                                                        Nutrient broth                                                                              No growth                                                       YM broth      Opaque growth, no pellicle or pigment,                                        becomes viscous on incubation                                   Salt tolerance in                                                                           Grows well with 0.4% NaCl, no                                   YM broth      growth with 0.6% NaCl.                                          Temperature range                                                                           10-35° C., optimum 20-30° C.                      pH range in YM broth                                                                        pH 3.0 to pH 10.0, optimum pH 4.0-10.0                          pH range in Nutrient                                                                        will show weak growth at pH 8.0 but                             broth         not below (after 13 days).                                      ______________________________________                                    

Atmospheric nitrogen fixed in nitrogen deficient media, molybdenum isrequired for nitrogen fixation.

In neutral or alkaline media, acid is produced. In very acid media, analkaline substance is produced which increases the pH of the medium.

    ______________________________________                                        Biochemical Characteristics                                                   ______________________________________                                        Catalase                 positive                                             Oxidase                  positive                                             Methyl Red Test          negative                                             Indol formation          negative                                             Urease                   positive                                             Amylase                  negative                                             Gelatin liquefaction     Negative                                             Nitrate reduction        positive                                             Citrate utilisation      negative                                             Acetylmethylcarbonyl formation                                                                         positive                                             CVP test                                                                      H.sub.2 S formation (from thiosulphate)                                                                negative                                             Cellulase                positive                                             ______________________________________                                        Carbohydrate Utilisation                                                                                        Incubation                                            Growth    Acid Production                                                                             Time                                        ______________________________________                                        D-glucose Positive  Positive      20 days                                     D-mannose Positive  Positive      13 days                                     D-galactose                                                                             Positive  Positive      20 days                                     D-fructose                                                                              Positive  Positive      13 days                                     D-arabinose                                                                             Positive  Positive      13 days                                     D-xylose  Negative  Negative      20 days                                     L-rhamnose                                                                              Negative  Negative      20 days                                     D-sucrose Positive  Positive      13 days                                     adonitol  Negative  Negative      20 days                                     D-maltose Negative  Negative      20 days                                     D-trehalose                                                                             Negative  Negative      20 days                                     D-lactose Negative  Negative      20 days                                     D-cellobiose                                                                            Negative  Negative      20 days                                     D-melibiose                                                                             Positive  Positive      13 days                                     dextrin   Negative  Negative      20 days                                     D-raffinose                                                                             Positive  Weak Positive 20 days                                     salicin   Negative  Negative      20 days                                     D-mannitol                                                                              Positive  Weak Positive 20 days                                     Na-alginate                                                                             Negative  Negative      20 days                                     ______________________________________                                        Effect of aminoacids on pigmentation & growth                                 (Burk's + glucose 7 days 27° C.)                                       ______________________________________                                        DL-alanine       no growth                                                    DL-serine        no growth                                                    DL-phenylalanine No growth                                                    DL-tyrosine      No growth                                                    DL-methionine    No growth                                                    L-cystine        No growth                                                    DL-tryptophan    No growth                                                    L-proline        growth, cream, glistening                                    L-hydroxyproline good growth, cream, glistening                               DL-aspartic acid no growth                                                    L-glutanic acid  very good growth, cream                                      DL-histidine     no growth                                                    L-lysine         poor growth, off white                                       L-arginine       growth, cream                                                L-citrulline     good growth, cream gummy                                     ______________________________________                                    

The preparation of indican may be achieved by culturing theindican-producing micoorganism, e.g. B. indica ATCC 19361, either in abatch process or in a continuous process, according to establishedmicrobiological techniques. The fermentation medium must be one which isa nutrient for the strain, and in which the strain will successfullyproduce indican. A typical medium is given in Example 1 below, Burk'shigh phosphate medium, is based on a defined mixture of sucrose andsimple inorganic salts. A complex medium, such as nutrient broth ortryptone broth, is also suitable. The microorganism is capable of fixingnitrogen and so a nitrogen-free medium can be used, provided the cultureis sufficiently aerated. Alternatively, in addition to aeration, a fixednitrogen source such as ammonia can be included in the medium. Apartfrom these considerations, the medium should contain all the essentialfactors needed for growth and polysaccharide production, includingsources of carbon and energy such as sucrose, phosphorus, magnesium andtrace elements. The culture may, as indicated above, be batchwise orcontinuous. Batchwise working is, by its nature, intermittent andcapital intensive and commercial production favours continuous working.

The indican can be isolated from the culture by precipitation, e.g. witha water-miscible solvent such as isopropanol. Because of the relativelyhigh solubility of indican in aqueous organic solvents, a higherproportion of precipitant is needed than for, say, xanthan.Alternatively, the suspended matter in the culture, e.g. cells, can beremoved by filtration or centrifugation and the polysaccharide obtainedby removing the water, e.g. by free-drying.

The following examples illustrate the invention further.

EXAMPLE 1 Production of indican from B. indica ATCC 19361

100 ml of sterile growth medium (Table 3) in a 500 ml conical flask wasinoculated with the Beijerinckia indica ATCC 19321 from a stock culturemaintained on growth medium solidified with agar (1.3%).

The flask was incubated at 30° C. for 10 days with shaking on a gyrotaryshaker (150 rpm).

The shake-flask culture was used to inoculate 4 l of growth medium in a5 l capacity stirred tank fermentation vessel. The culture wasmaintained at 30° C., aerated at 2 l min⁻¹ and stirred at an impellerspeed of 400 rev min⁻¹. After 100 h, the contents of this vessel wastransferred aseptically into 40 l of culture medium in a 50 l capacitystirred-tank fermentation vessel. The culture was maintained at 30° C.,aerated at 10 l min⁻¹ and stirred at 100 rev. min⁻¹. As the fermentationproceeded and the viscosity increased, the impeller speed was increasedto 150 rev min⁻¹ 88 h after inoculation and to 210 rev min⁻¹ 102 h afterinoculation. 117 h after inoculation, the apparent viscosity of theculture broth had increased to 12,000 cp at a shear rate of 1 sec⁻¹ asmeasured on a Wells-Brookfield cone and plate microviscometer. At thistime, the fermentation broth was added with mixing to isopropanol (4volumes). The mixture was left until a distinct precipitate formed (8days). The precipitate was then collected, pressed in muslin to removeexcess liquor and freeze dried to yield 200 g of product.

                  TABLE 3                                                         ______________________________________                                        Culture Medium                                                                Component         Concentration (gl.sup.-1)                                   ______________________________________                                        Sucrose           20                                                          K.sub.2 HPO.sub.4 0.64                                                        KH.sub.2 PO.sub.4 0.16                                                        MgSO.sub.4 . 7H.sub.2 O                                                                         0.2                                                         NaCl              0.2                                                         CaCl.sub.2 . 2H.sub.2 O                                                                         0.043                                                       Na.sub.2 MoO.sub.4                                                                              1 × 10.sup.-3                                         FeSO.sub.4 . 7H.sub.2 O                                                                         3 × 10.sup.-3                                         H.sub.3 BO.sub.4  3 × 10.sup.-3                                         CoSO.sub.4 5H.sub.2 O                                                                           1.2 × 10.sup.-3                                       CuSO.sub.4 . 5H.sub.2 O                                                                         0.1 × 10.sup.-3                                       MnCl.sub.2 . 4H.sub.2 O                                                                         0.09 × 10.sup.-3                                      ZnSO.sub.4 . 7H.sub.2 O                                                                         1.2 × 10.sup.-3                                       ______________________________________                                    

The structure of the polysaccharide, indican, obtained was examined asfollows;

Indican (50 mg) was heated with 2 N sulphuric acid for 4 h at 105° C.The solution was neutralised by addition of excess barium carbonate, andthe supernatant which formed on centrifuging this mixture de-ionised bytreatment with Amberlite IR 120 (H⁺) and Amberlite 410 (Ac⁻) resins. Onconcentration, a syrup was obtained which showed spots on thin layerchromatograms using as the solvent n-BuOH:pyridine water (6:4:3 byvolume) corresponding to glucose and rhamnose. A portion of the syrupwas converted into the aldononitrile acetate derivatives by heatingfirst with hydroxylamine in pyridine and then with acetic anhydride. Gaschromatographic analysis of the derivatives showed the presence ofglucose, mannose and rhamnose in the proportions of 2 glucose: 1mannose: 1.6-1.8 rhamnose. N. B. The Kelco product Heteropolysaccharide7 from Azotobacter indicus var. myxogenes was found to contain glucoseand rhamnose only in a ratio of approximately 5 glucose: 2 rhamnose.

The presence of an acid component was demonstrated by decarboxylation.On boiling with hydrochloric acid (19% HCl) for 2 h, the carbon dioxideyield as measured by absorbtion in sodium hydroxide and titration ofexcess base with hydrochloric acid was 4.1% by weight of thepolysaccharide. This tends to imply an anhydrouronate content of about16%. From these results, it can be concluded that the repeating unit ofthe polysaccharide is composed of Glc, Man, Rha and an acidic componentin the approximate ratio 2:1:2:1. The acid fragment was isolated as asyrup by preparative paper chromatography (6:4:3 nBOH: pyridine: water,R_(G) 0.45-0.60) of an acid hydrolysate (IN H₂ SO4, 1 h at 100° C.) ofcell free indican. The very low colour yield obtained with this materialupon testing with the carbazole (Knutson, C. A. et al Anal.Biochem. 24,470 (1968)) and the phenol/sulphuric acid (Dubois, M. et al. Anal.Chem., 27 350 (1956)) reagents strongly indicated that the material wasnot a uronic acid. Methylation analysis of the cell-free indican gavepartially methylated alditol acetates consistent with the followingfragments being present in the repeating unit: 2 (→3) Glc; 1 (1→4) Man;at least 1 (1→4) Rha. The methylated polysaccharide was reduced (LiAlH₄in boiling tetrahydrofuran for 17 h) and the product was hydrolysed andthe resulting fragments were acetylated. A new peak was observed in thegas chromatogram of the acetylated mixture (stationary phase ECNSS-M(Phase Separations Ltd., Queensferry, N. Wales; and U.S. Pat. No. 3 263401) T_(TMG) =3.96). From the mass spectrum of the substrance causingthis new peak, it was deduced that the structure is as given in Formula(1) ##STR1## (where the numbers and broken lines indicate the massspectral fragmentation).

A larger sample of the acidic fraction was obtained and analysed asfollows.

The indican sample (6 g) was refluxed with 2 N hydrochloric (100 ml)acid for 7 h. The reaction mixture was cooled and treated with activatedcharcoal (0.5 h), and filtered through a fluted filter paper. Thecharcoal residue was thoroughly washed with water and then absoluteethanol and the filtrate and the washings were combined and concentratedby rotary evaporation. The resulting syrup was redissolved in water (30ml) and re-concentrated (x3) to effect removal of residual hydrochloricacid. The mixture thus obtained was heated for 30 min at 60° C. withtriethylamine (2 ml) in water (50 ml) and the cooled solution applied toa column of Amberlite IRA 410 resin (acetate form, 600 ml) and elutedwith water (2x). The acidic components of the hydrolysate were recoveredfrom the resin by washing with 20% acetic acid (900 ml). This solutionwas concentrated to a syrup which was fractionated by preparative paperchromatography (Whatman 3 MM, n-butanol:pyridine:water 6:4:3 by volume)into three distinct fractions. The fastest running material (detected byheating the paper strips after spraying with p-anisidine hydrochloridein n-butanol) had a mobility relative to that of mannose (R_(M)) of1.30, and co-chromatographed with rhamnose. The second fraction hadR_(M) 0.87, in close agreement with the mobility reported (N. K.Kochetkov, et al Carbohydr Res 51 (1976) 229-237) for3-O-[(R)-1-carboxyethyl]-L-rhamnose in this solvent system (R_(M) 0.97).The slowest moving spot appeared at R_(M) 0.42 consistent with themobility expected for an acidic odisaccharide.

The detection of the above-mentioned reduced methylated derivative andthe above-described chromatographic analysis imply that the acidfragment in the repeating unit is a 6-deoxyhexose substituted withlactic acid at either O or O-4 via an ether linkage, and linked to theremainder of the polysaccharide chain through O-4 or O-3 respectively.

Acetyl ester substitution in the polysaccharide was demonstrated by thefollowing experiment. An aqueous solution of indican (500 ml 0.2% w/v)was made alkaline (pH 12) by addition of 10 N sodium hydroxide. Afterstanding at room temperature for 1 hr., concentrated sulphuric acid wasadded dropwise to lower the pH to 2. This solution was fractionallydistilled and the distillate (about 200 ml) after stirring withAmberlite IR120(Na⁺) resin was concentrated to give a white solid. Theproduct was converted into the crystalline S-benzyl thiourea salt; thiscompound had a melting point (135° C.) identical with an authenticsample of the SS-benzyl thiourea salt of acetic acid. No depression ofmelting point occurred on admixture of the two. The acetyl content ofindican was estimated by the hydroxamic acid/ferric ion complex methodto be 12-15% of the polysaccharide.

A sample of purified, essentially cell-free indican prepared asdescribed in Example 4 had a negative specific optical rotation (-61.2°)and a film formed from this material had strong absorbances at 3390,1735, 1615, 1375, 1250 and 1050 cm⁻¹ in the infra-red spectrum.Periodate oxidation studies on de-acetylated cell-free indican showedthe consumption of 3-5 moles of periodate per repeating unit, consistentwith the structure:

    ______________________________________                                        1 → 3   D-glucose (2 moles)                                            1 → 4   Mannose (1 mole)                                               1 → 4   Rhamnose (1 or 2 moles)                                        1 → 3 or 4                                                                            0-(1-carboxyethyl)-rhamnose                                                   (1 mole).                                                      ______________________________________                                    

The glucose was shown to be the D-enantiomer by reaction of neutralised,hydrolysates with D glucose-oxidase.

The polysaccharide indican is found to possess an inherent viscosity ofabout 33.5 dl/g. A useful discussion of viscosity measurement of asubstance such as aqueous indican is given by C. Tanford in PhysicalChemistry of Macromolecules, published by Wiley & Sons, 1961. For thepurposes of this specification, inherent viscosity is taken as thefunction ##EQU1## where c is the concentration in g/100 ml and η and η'are the viscosities of the solution at concentration c and the solventrespectively, measured at 25° C. in a μ-tube viscometer. As c tends tozero, the inherent viscosity approaches the intrinsic viscosity [η].Measurements for the figure quoted are taken at c=0.01 g/l FIGS. 1 and 2in the accompanying drawings illustrate the viscosity characteristics of1% by weight solutions of indican. FIG. 1 represents the effect of shearstress on shear rate in a 1% aqueous solution, the hysteresis indicatingclearly the thixotropic nature of the gel. FIG. 2 is a plot of anequilibrium flow curve of viscosity against the shear rate for a 1%solution in 1% aqueous NaCl, showing thinning with increasing shear.

The polysaccharide produced by Beijerinckia indica (Starket and De) wasfound by J. K. N. Jones, M. B. Perry and W. Sowa, (Canad. J. Chem. 412717-2715) and V. M. Parikh and J. K. N. Jones (Canad. J. Chem. 412826-2835, 1963) to be quite different from that described above. Theyclaimed the polymer to be liner molecule composed of repeating units ofD-glucuronic acid, D-glucose and D-manno-heptose. Later workers, Haug etal., found the uronic acid to be guluronic not glucuronic acid.

EXAMPLE 2 Xanthan Suspension

A solution of the isopropanol-precipitated matter from a culture of B.indica ATCC 19361, according to Example 1, was dissolved in methanol toform a 0.8% w/v solution. This solution was diluted with ethanol (0.6volume) to give a mixture containing 0.5% polysaccharide 37% ethanol and62.5% methanol. To this gel was added dry xanthan gum to give a mixturewith a 20% total solids content. On standing, a smooth gel formed whichshowed no tendency for the solid to separate out or for syneresis tooccur for a period greater than three months. A similar compositioncontaining no indican separated into a liquid and a solid sedimentwithin hours of mixing. The indican-containing preparation dispersed andhydrated rapidly when stirred into water.

EXAMPLE 3 Premium Quality Flat White Paint

The following procedure was carried out, namely the formation of apremix to which were added pigments and resin.

    ______________________________________                                                                 lbs./106.6                                                                    gals                                                 ______________________________________                                        Premix: (added with vigorous mixing at 1800 rpm.)                             water                      47.0                                               potassium tripolyphosphate 1.5                                                tamol 850 (30% solids) (dispersant)                                                                      6.7                                                indican (0.7% soln.)       130.0                                              ethylene glycol            15.0                                               hexylene glycol            35.0                                               nopco NXZ (antifoaming agent)                                                                            2.0                                                The following were then added at 1800 rpm. then                               the speed was increased to 3500 rpm. and the mixture was                      ground 20 minutes:                                                            titanium dioxide pigment   300.0                                              china clay                 75.0                                               quartz (mean particle size 9 microns)                                                                    70.0                                               Let-down: (added at low speed mixing)                                         acrylic latex emulsion (Rhoplex AC-22)                                        (46.5% solids)             470.0                                              water                      15.0                                               bis(phenylmercury)dodecenylsuccinate                                                                     1.0                                                nopco NXZ                  1.0                                                indican (0.7% soln.)       57.0                                                                          1226.5                                             ______________________________________                                    

Adjust to pH 9.5 with 28% ammonia

EXAMPLE 4 Cell-free Indican

A culture of Beijerinckia indica ATCC 19361 on an agar slope wastransferred aseptically with 5 ml of Ringers solution into a shake-flaskcontaining 200 ml of sucrose-Burk's medium. After shaking on an orbitalshaker at 35° C. for 10 days, the culture had become viscous. It wasdiluted with three volumes of distilled water and heated at 65° C. for 1h. The hot solution was centrifuged for 1 h at about 20,000 g toseparate the cellular matter. The clear supernatant, on freeze dryinggave 0.4 g white fibrous polysaccharide which on dissolution gave aclear, colourless and viscous solution. The cell-free sample of Indicanthus prepared had [α]_(D) ²⁰ -61.2° (c 0.216 in water).

EXAMPLE 5

    ______________________________________                                        Gelled Pet Food                                                               ______________________________________                                        meat (chunks)           46%                                                   water                   52%                                                   benzoic acid            0.10%                                                 sodium metabisulphite   0.15%                                                 indican                 1%                                                    ______________________________________                                    

The partially cooked meat chunks were dispersed in the 2% aqueoussolution of indican and heated for 10 min at 80° C. The benzoic acid andsodium metabisulphite were added and the blend was allowed to cool toform a gelled pet food.

EXAMPLE 6 Paint Stripper

A solution of indican in 1:1 methylene chloride:methanol (0.5% byweight) was formed by diluting a 1% methanolic solution of Indican withmethylene chloride. The solution was a white, translucent gel which hada texture very suitable for painting onto and adhering to verticalpainted surfaces. The solution has the same effectiveness as ahydroxyalkylcellulose-thickened formulation.

EXAMPLE 7 Oil/Water Emulsion

A 0.5% by weight solution of indican in water (100 parts by weight) hasadded thereto 50 parts by weight of kerosene and the mixture ishomogenised. A stable emulsion is obtained. Similarly stability isachieved with salad oil.

EXAMPLE 8 Film

A 1.0% by weight solution of indican in warm water optionally containingglycerol as a plasticiser was degassed and run over apolytetrafluoroethylene sheet and dried for several hours at about 50°C. The resulting film was stripped from the sheet to give a flexible,water-swellable, biodegradable film.

The infrared spectrum of a thin film of indican (without plasticiser)contains a pronounced carbonyl peak at 1730 cm⁻¹, which is assigned tothe carbonyl of the ester groups (acetate) and a lesser peak at 1610cm⁻¹, which is assigned to the carbonyl of the carboxymethyl groups.

EXAMPLE 9 Liquid Membranes

A water in oil emulsion was made by high shear mixing (Silverson mixer)water (1 volume) into liquid paraffin (3 volumes) containing 2% w/v Span85. This emulsion (4 volumes) was dispersed into an aqueous solution (3volumes) of indican (0.5% w/v) using mild agitation (Citenco mixer) togive a smooth water-in-oil-in-water three phase system. Upon standing,the outer thixotropic aqueous phase developed structure which preventedcoalescence or separation of the dispersed emulsion. The three phasesystem could be easily poured and remained visually unchanged afterstanding for 1 month at room temperature.

Chemicals, such as those used in the pharmaceutical or agriculturalindustries, could be incorporated into the internal aqueous phase astaught in U.S. Pat. No. 4,083,798.

The advantages of indican-containing formulations of this kind are thatthey are simpler to prepare, have improved flow characteristics and theuse of expensive gelling polysaccharides (e.g. agar) is obviated.

EXAMPLE 10 Ethylene glycol and other non-aqueous solvent gels

A 1% w/v solution of indican in ethylene glycol was prepared by allowingthe solid to swell for several hours in the solvent, and then applyingagitation. This solution was extremely viscous and appeared to be a weakgel. The gel-like characteristics were increased by heating at 100° C.for 10 min. and allowing the solution to cool. The gel remainedunchanged upon standing in the atmosphere for 1 year. Neither was itaffected by refrigeration to -20° C.

These properties are of interest in controlling the release of an activecompound (e.g. perfume or insect pheromone) or in the preparation of alubricating mixture.

Similarly gels can be produced in methanol, methanol mixed withmethylene dichloride (see Example 6), methanol/ethanol mixtures (seeExample 2), and also in ethanol, propan-2-ol, and acetone eachcontaining small (10-20% by volume) proportions of water.

We claim:
 1. Indican, a polysaccharide consisting essentially of (1→3)glucose, (1→4) mannose, (1→4) rhamnose and (1→3 or4)-O-(1-carboxyethyl)rhamnose units in a molar ratio of about 2:1:1-2:1respectively; containing 12-15% by weight acetyl units; [α]_(D) ²⁰ about-61°; having principle absorption bands in the infra red band at 3390,1735, 1615, 1375, 1250 and 1050 cm⁻¹ ; a solubility of at least 1% byweight in methanol and in ethylene glycol; and an inherent viscosity ofabout 33.5 dl/g; separated from the culture from which it was prepared.2. Substantially cell-free indican obtained from a culture ofBeijerinckia indica ATCC
 19361. 3. A flexible film of indican formed bytaking a thin layer of an indican solution and evaporating the solvent.